Supporting Information Table of Contents
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1 Supporting Information Table of Contents Supporting Information Figure 1 Page 2 Supporting Information Figure 2 Page 4 Supporting Information Figure 3 Page 5 Supporting Information Figure 4 Page 6 Supporting Information Figure 5 Page 7 Supporting Information Figure 6 Page 8 Supporting Information Figure 7 Page 9 Supporting Information Figure 8 Page 10 Supporting Information Figure 9 Page 11 Supporting Information Figure 10 Page 12 Supporting Information Figure 11 Page 13 Supporting Information Figure 12 Page 14 Supporting Information Figure 13 Page 15 Supporting Information Table 1 Page 16 1
2 Supporting Information Figure 1. Carabin gene targeting. (A) Schematic representation of the targeted region of murine Carabin locus (Carabin +), targeting construct, targeting allele, Neo-deleted and exons 4-5 floxed (Carabin f) allele, and Neo-deleted and exons 4-5 deleted (Carabin -) allele. Filled boxes represent exons; filled triangles, LoxP sites. Note that the NeoR cassette is flipped and floxed (FRT sites are not represented) (B) Screening of Carabin f/-, Carabin +/-, Carabin +/+ and Carabin -/- mouse line progenies by PCR on tail DNA, using primers 1 (Lf1), 2 (Ef) and 3 (Er1); see Generation of ES cells, Carabin -/-, and conditional Carabin -/- mice in the Methods section. Filled box represents exons; filled triangles, LoxP sites. Primers 1 and 3 reveal a 1.15 kb floxed (Carabin f), a 0.99 kb WT 2
3 (Carabin +) and a 0.42 kb deleted (Carabin -) alleles, respectively. Primers 2 and 3 reveal a 0.20 kb WT and 0.32 kb floxed alleles, respectively. (C) Quantitative real-time RT-PCR analysis of Carabin mrna expression in splenocytes from Carabin +/+ or Carabin -/- mice, using three different probes matching exons 2-3 (Probe 1), exons 4-5 (Probe 2), exons 6-7 (Probe 3). Each sample was normalized to the endogenous control Hprt1. (D) Western blot analysis of splenic and thymic cells from Carabin +/+ or Carabin -/- mice. Actin was used as loading control. (E) PCR analysis of Carabin deletion. Carabin f and Carabin - alleles were amplified from genomic DNA of purified splenic total B or T cells (depending on the conditional KO model), and thymocytes from Carabin f/- mice expressing Cre (+) or not (-), using a combination of primers 1, 2, and 3 (depicted in Supplementary Figure 1b). (F) Immunoblot analysis of Carabin expression in purified splenic total B or T cells, and thymocytes from Carabin f/- mice expressing Cre (+) or not (-), using an anti-carabin antibody. Actin was used as loading control. (C; errors bars, standard deviation). 3
4 Supporting Information Figure 2. Serum Ig production in Carabin -/- mice. Sera from Carabin +/+ and Carabin -/- mice were collected and total IgG, IgM, IgG1, IgG2b, and IgG3 were determined by ELISA (microgram / milliliter). Each point represents the result for one animal. The results of two-tailed Mann-Whitney test are indicated. 4
5 Supporting Information Figure 3. Increased response of Carabin-deficient T and B Cells. (A) Flow cytometry analysis of cell surface expression of CD69, CD44 and CD25 on Carabin +/+ and Carabin -/- CD4 + T cells after stimulation for 72h with anti-cd3 antibody (2 µg/ml), anti-cd3+anti-cd28 antibodies (2 µg/ml each), or medium alone. (B) Flow cytometry analysis of Erk phosphorylation in Carabin +/+ and Carabin -/- CD4 + T cells after stimulation for 10 min with anti-cd3 antibody (2 µg/ml) or with medium alone. (C) Flow cytometry analysis of B220+ B cell surface expression of CD86, CD69 and MHCII on Carabin +/+ and Carabin -/- CD19 + B cells after stimulation for 72h with LPS (10 µg/ml) anti-igm antibody (10 µg/ml), or medium alone and (D) Flow cytometry analysis of Erk phosphorylation in Carabin +/+ and Carabin -/- B220 + B cells after stimulation for 10 min with anti-igm antibody (10 µg/ml), or with medium alone. (A-D) Data correspond to three 5
6 independent experiments. Errors bars: standard deviation. The results of two-tailed Mann- Whitney test are indicated. 6
7 Supporting Information Figure 4. Erk phosphorylation is enhanced in Carabin -/- T cells after stimulation with anti-cd3 and anti-cd3 / anti-cd28 antibodies. Flow cytometry analysis of Erk phosphorylation in Carabin +/+ and Carabin -/- CD4 + T cells after stimulation for 10 min with anti-cd3 antibody (2 µg/ml) (dashed line), anti-cd3+anti-cd28 antibodies (2 µg/ml each) (solid line), or with medium alone (shaded gray). Numbers indicate Mean Fluorescence Intensity. Data are representative of three independent experiments. The corresponding statistical analysis is represented in Supporting Information Fig 3B. 7
8 Supporting Information Figure 5. Ig levels (nanogram/milliliter) were analyzed in supernatants from splenic cell cultures, after 72h of stimulation with LPS (10 µg/ml) (in this case, IgM, IgG, IgG2b, IgG3 were quantified) or LPS (10 µg/ml) + IL-4 (20 ng/ml) (in this case, IgG1 were quantified). Each point represents the result for one animal. The results of two-tailed Mann-Whitney test are indicated. 8
9 Supporting Information Figure 6. Intracellular Ca 2+ response of Indo-1 loaded splenic purified (CD43-negative) B cells from Carabin +/+ (orange and red lines) and Carabin -/- (blue and green lines) mice. Data were collected for 120s to establish baseline violet/blue ratios; cells were then stimulated by the addition of 5 μg/ml anti-mouse IgM (A) or 1 M ionomycin (B) and data were collected for 5 additional minutes. 9
10 Supporting Information Figure 7. Carabin deficiency speeds up the production of anti- OVA IgM. Six- to eight-week-old mice of the indicated genotype were injected intraperitoneally with 100 μg OVA in complete Freund s adjuvant and bled seven days after injection. Anti-OVA IgM titers were determined by ELISA. Each point represents the result for one animal. The results of two-tailed Mann-Whitney test are indicated. Because the results obtained in Carabin +/+ and in Carabin f/- Cre-negative mice were not different, these animals were pooled in the same control group. 10
11 Supporting Information Figure 8. Carabin deficiency did not modify germinal center kinetics in OVA-immunized mice. (A,B) Flow cytometry analysis of OVA-specific B cells (B220 + /OVA + ) (left) and germinal center B cells (CD95 + /GL7 + ) in OVA-specific population (middle, flow cytometry plots; right: the histograms show percentages of cells) in spleen (A) and lymph nodes (B), of Carabin -/- and Carabin +/+ mice, at day 7 after immunization with 100 μg OVA in complete Freund s adjuvant. (A, B) Data are representative of three independent experiments. (A, B; errors bars, standard deviation). 11
12 Supporting Information Figure 9. Increased early antigen-specific T-independent B cell response in Carabin KO mice. (A, B) 12-week-old mice of the indicated genotype were injected intraperitoneally with 100 μg NP-LPS in PBS and bled seven days after injection. Anti-NP (A) IgM or (B) IgG titers were determined by ELISA. Each point represents the result for one animal. The results of two-tailed Mann-Whitney test are indicated. (C, D) AntidsDNA (C) anti-thyroglobulin (D) IgG titers were determined by ELISA in sera of Carabin +/+ (n=3) and Carabin -/- (n=3) mice. (C, D; errors bars, standard deviation). 12
13 Supporting Information Figure 10. B cell response in Carabin deficient mice after infection with Borrelia burgdorferi. Carabin -/- (n=3) and control (n=3) mice were injected with 10 6 Borrelia burgdorferi (Bb) spirochetes. The anti-bb specific IgG response was checked at the indicated times, by ELISA. The results represent the optical densities for a 1/50 serum dilution. (Errors bars, standard deviation). 13
14 Supporting Information Figure 11. (A, B) Eight to ten-week-old mice of the indicated genotypes were treated with 40 μg of CpG intraperitoneally every other day for 2 weeks. Serum was collected before treatment (day 0), and at day 7, 14, 28, 49 after the first injection. Anti-thyroglobulin (A) and anti-actin (B) IgG titers were determined by ELISA (errors bars, standard deviation). (A, B) Data are representative of three independent experiments. 14
15 Supporting Information Figure 12. Flow cytometry analysis of cell surface expression of CD44 on Carabin +/+ and Carabin -/- splenic purified (CD43-negative) B cells after stimulation with anti-igm (2 µg/ml), CpG-DNA (1 µg/ml), or anti-igm (2 µg/ml) plus CpG- DNA (1 µg/ml) for 6h (A) and 12h (B) in vitro. Bars represent the increase of CD44 MFI on stimulated cells versus unstimulated cells (errors bars, standard deviation). Data correspond to four independent experiments. The increases of CD44 MFI on Carabin +/+ and Carabin -/- B cells were compared between anti-igm plus CpG-DNA stimulated cells and CpG-DNA stimulated cells using a Yates' continuity corrected Chi-square test. 15
16 Supporting Information Figure 13. Proposed scenario of the integration of Carabin in BCR and TLR9 signaling. BCR (B-cell Receptor); TLR9 (Toll-like receptor 9); PLCγ (Phospholipase Cγ); PIP (phosphatidylinositol phosphate); DAG (Diacylglycerol); IP-3 (Inositol trisphosphate); PKC (Protein Kinase C). 16
17 Carabin +/+ Carabin -/- Total cellularity (x10 6 ) Spleen 112 ± ± 35 Lymph nodes 4.8 ± ± 3.5 Spleen (% and absolute numbers) B cells 50.4% ± % ± 9.3 ( ± 15.9) ( ± 12.9) Transitional % ± % ± 4.8 ( ± 4.7) ( ± 9.7) Transitional 2 3.5% ± % ± 0.8 ( ± 1.32) ( ± 0.6) Follicular B cells 68.9% ± % ± 4.7 ( ± 17.6) ( ± 12.7) Marginal zone 9.6% ± % ± 3.5 ( ± 3.2) ( ± 3.6) CD4+ T cells 23.9% ± % ± 4.9 ( ± 6.0) ( ± 7.1) CD8+ T cells 15.5% ± % ± 4.9 ( ± 3.7) ( ± 4.3) Lymph nodes (% and absolute numbers) B cells 22.8% ± % ± 3.6 ( ± 0.8) ( ± 0.8) CD4+ T cells 43.7% ± % ± 1.2 ( ± 1.2) ( ± 1.5) CD8+ T cells 29.0% ± % ± 3.7 ( ± 0.7) ( ± 1.1) Bone marrow (%) ProB/Pre-B 12.6% ± % ± 5.5 Immatures 4.5% ± % ± 2.5 Transitional 1.0% ± % ± 0.4 Recirculating mature 6.2% ± % ± 1.8 Thymus (%) CD4+ T cells 9.2% ± % ± 0.4 CD8+ T cells 4.3% ± % ± 0.6 DN 2.1% ± % ± 1.1 DP 84.3% ± % ± 2.2 Supporting Information Table 1. Cellularity of lymphoid organs and respective subpopulations in Carabin KO mice. Quantitation of lymphoid populations in the indicated tissues from the indicated genotypes of 12-week-old mice. The total cellularity of spleen and 17
18 lymph nodes is shown in the top panel. B cell subpopulations were identified by flow cytometry with the following markers: B cells (B220 + IgM + ); Pro/PreB (B220 + IgM - ); Immature (B220 med IgM + ); recirculating mature (B220 high IgM + ); transitional 1 (IgM + CD23 - CD21 - ); transitional 2 (IgM + CD23 + CD21 high ); follicular (IgM + CD23 + CD21 low ) and marginal zone (IgM + CD23 - CD21 high ). DN: double-negative; DP: double-positive T cells. Means and standard deviation of six Carabin -/- and seven Carabin +/+ mice are shown. 18
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